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. Author manuscript; available in PMC: 2013 Sep 9.

Published in final edited form as: Pediatr Res. 2012 Nov 30;73(3):252–262. doi: 10.1038/pr.2012.177

Figure 7a: ET-1 increases rho-kinase activity in normal and PPHN fetal PAECs. Effect of ET-1 treatment (100nM) on rho-kinase activity was measured by MYPT-1 and phosphorylated-MYPT-1 (p-MYPT-1) protein by western blot analysis in normal and PPHN PAEC whole cell lysates (N=4 clones). Rho-kinase activity as measured by p-MYPT-1:MYPT-1 protein ratio increased by 44% in normal and 55% in PPHN PAECs with ET-1 treatment (p<0.01 for each comparison).

Figure 7b: ET-1 SiRNA decreases rho-kinase activity in normal and PPHN PAECs. The effect of ET-1 SiRNA (4μM) on rho-kinase activity was measured by MYPT-1 and phosphorylated-MYPT-1 (p-MYPT-1) protein by western blot analysis in normal and PPHN PAEC whole cell lysates (N=4 clones). p-MYPT-1:MYPT-1 protein ratio decreased by 35% in normal and PPHN PAECs with ET-1 SiRNA treatment (p<0.01 for each comparison).

Figure 7c: Rho-kinase inhibition prevents the decreases in 3D tube formation by normal PAECs and rescues the abnormal in vitro PPHN phenotype after ET-1 treatment. 3D tube formation was assessed in normal and PPHN PAECs in the presence and absence of ET-1 treatment (100nM) with and without y-27632 (1μM) (rho-kinase inhibitor). After ET-1 treatment y-27632 increased tube length by 25% (p<0.05)(fig not shown) and branch points/HPF by 32% (p<0.01) in normal PAECs restoring tube formation to normal. In PPHN PAECs, y-27632 increased tube length by 75% (p<0.01)(fig not shown) and branch points/HPF by 95% (p<0.01) restoring tube formation to values seen in normal PAECs.

Figure 7a: ET-1 increases rho-kinase activity in normal and PPHN fetal PAECs. Effect of ET-1 treatment (100nM) on rho-kinase activity was measured by MYPT-1 and phosphorylated-MYPT-1 (p-MYPT-1) protein by western blot analysis in normal and PPHN PAEC whole cell lysates (N=4 clones). Rho-kinase activity as measured by p-MYPT-1:MYPT-1 protein ratio increased by 44% in normal and 55% in PPHN PAECs with ET-1 treatment (p<0.01 for each comparison).

Figure 7b: ET-1 SiRNA decreases rho-kinase activity in normal and PPHN PAECs. The effect of ET-1 SiRNA (4μM) on rho-kinase activity was measured by MYPT-1 and phosphorylated-MYPT-1 (p-MYPT-1) protein by western blot analysis in normal and PPHN PAEC whole cell lysates (N=4 clones). p-MYPT-1:MYPT-1 protein ratio decreased by 35% in normal and PPHN PAECs with ET-1 SiRNA treatment (p<0.01 for each comparison).

Figure 7c: Rho-kinase inhibition prevents the decreases in 3D tube formation by normal PAECs and rescues the abnormal in vitro PPHN phenotype after ET-1 treatment. 3D tube formation was assessed in normal and PPHN PAECs in the presence and absence of ET-1 treatment (100nM) with and without y-27632 (1μM) (rho-kinase inhibitor). After ET-1 treatment y-27632 increased tube length by 25% (p<0.05)(fig not shown) and branch points/HPF by 32% (p<0.01) in normal PAECs restoring tube formation to normal. In PPHN PAECs, y-27632 increased tube length by 75% (p<0.01)(fig not shown) and branch points/HPF by 95% (p<0.01) restoring tube formation to values seen in normal PAECs.